Wnt antagonist genes in kidney tumor progression and metastasis. Main goal of this project is to investigate the role of Wnt antagonist genes in the progression and metastasis of renal cancer. The rationale is that Wnt antagonist genes are inactivated through CpG methylation pathways with the result that Wnt/ b-catenin pathways are activated and induce malignant transformation of various organs. However, such studies are lacking in kidney cancer. Three specific hypotheses will be tested to determine if: 1) Inactivation of Wnt antagonist genes is involved in the progression and metastasis of kidney cancer. 2) The mechanisms of inactivation of the Wnt antagonist genes are through epigenetic pathways such as DNA methylation, histone modification and chromatin remodeling. 3) Transfection of Wnt antagonist genes suppresses the in vitro and in vivo growth and metastasis of kidney cancer. To test these hypotheses, we will pursue the following specific aims. Specific Aim # 1: To investigate whether inactivation of Wnt antagonist genes is involved in the progression and metastasis of human renal cell carcinoma. Based on the preliminary data, we have screened several Wnt antagonist genes and have identified six genes that are silenced in kidney cancer. These genes are: secreted frizzled-related protein-1 (sFRP-1), sFRP-2, sFRP-4, sFRP-5, Wnt inhibitory factor-1 (Wif-1) and DICKKOPF-3 (DKK-3). Under this specific aim, we will determine the levels of mRNA and protein expression of Wnt antagonist genes in normal and different stages and grades of kidney cancer. The mRNA expression will be analyzed by real-time RT- PCR and protein expression by immunohistochemistry and Western blotting. Specific Aim # 2: To investigate the mechanisms of inactivation of Wnt antagonist genes in kidney cancer. Under this aim, we will analyze hypermethylation of CpG Islands in promoter regions of Wnt antagonist genes using sodium bisulfite methylation techniques and confirm by direct DNA sequencing. We will also investigate whether the mechanisms of inactivation of Wnt antagonist genes are due to DNA methyltransferase (DNMT-1, DNMT3a, DNMT3b), demethylase (MBD2) genes, histone acetylation and chromatin remodeling through analysis of these parameters in kidney cancer tissues. Specific Aim # 3: To investigate the functional role of Wnt antagonist genes in kidney cancer. Under this specific aim, we will transfect full-length Wnt antagonist gene cDNA in dominant-negative kidney cancer cells and establish the stable transfectant cells. We will analyze in vitro and in vivo growth of transfected kidney cancer cells. Also analyze in vitro invasiveness (extra-cellular matrix binding assay, invasion assay and soft agar colony forming efficiency) of transfected and parental cells. Successful completion of these experiments will demonstrate the functional role of Wnt antagonist genes in the suppression of kidney cancer growth, progression and metastasis and also the mechanism of inactivation of these genes in kidney cancer. In the future, these results may provide better strategies for the management of kidney cancer progression and metastasis. Renal cell carcinoma (RCC) is the third most common malignancy of the genitourinary system. Based on the published literature, it is clear that Wnt antagonist genes are associated with various cancers. However, such studies are lacking in kidney cancer. We have proposed to investigate the functional role of Wnt antagonist genes in the progression and metastasis of renal cell carcinoma using both in vitro and in vivo models. Successful completion of proposed experiments may provide us with the better strategies for the management of kidney cancer.